UNIVERSITY   OF   CALIFORNIA 
COLLEGE    OF  AGRICULTURE 
AGRICULTURAL    EXPERIMENT   STATION 
BERKELEY,   CALIFORNIA 


ALTERNARIA  ROT  OF  LEMONS 


E.  T.  BARTHOLOMEW 


BULLETIN  408 

October,  1926 


UNIVERSITY  OF  CALIFORNIA  PRINTING  OFFICE 

BERKELEY,  CALIFORNIA 

1926 


Digitized  by  the  Internet  Archive 

in  2012  with  funding  from 

University  of  California,  Davis  Libraries 


http://www.archive.org/details/alternariarotofl408bart 


ALTERNARIA   ROT  OF  LEMONS 

E.  T.  BARTHOLOMEW2 


INTRODUCTION 

With  the  possible  exception  of  Penicillium,  Alternaria  is  probably 
instrumental  in  causing  more  California  lemons  to  decay  than  any 
other  known  fungus.  That  the  same  may  be  true  for  other  lemon- 
producing  localities  is  indicated  by  the  following  quotation:  "In  six 
cars  of  lemons  from  Italy,  from  4  to  55%  or  an  average  of  18%  of 
Alternaria-inf ected  lemons  was  found. '  '3  The  same  article  states  that 
while  this  survey  was  being  made  ninety-one  cars  of  lemons  from 
California  showed  an  average  of  10  per  cent  of  Alternaria  rot. 

In  this  connection  it  is  of  interest  that  Fawcett4  found,  as  a  result 
of  a  study  of  the  lemons  shipped  to  the  central  and  eastern  markets  of 
the  United  States  during  the  latter  part  of  July  and  in  August,  1924, 
that  under  the  conditions  prevailing  at  that  time  Alternaria  was 
responsible  for  more  decay  than  all  of  the  other  fungi  combined. 

Alternaria  rot  develops  in  lemon  fruits  almost  exclusively  under 
storage,  transit  or  market  conditions.  It  appears  in  the  unpicked  fruit 
only  when  it  is  allowed  to  become  over-ripe  or  when  some  abnormal 
condition,  such  as  a  freeze,  has  materially  weakened  the  fruit.  As 
will  be  shown  later  on  in  this  paper,  hundreds  of  tests  have  proved 
that  practically  every  fruit  is  potentially  infected  by  the  time  it  is  a 
few  weeks  old ;  but  the  development  of  the  fungus  and  the  destruction 
of  the  fruit  is  delayed  by  the  vitality  of  the  fruit. 

During  the  early  part  of  this  study  decaying  fruits  were  obtained 
from  a  large  number  of  the  lemon  packing  houses  in  southern  Cali- 
fornia and  a  few  from  the  central  and  northern  parts  of  the  state. 
These  fruits  were  supposed  to  be  infected  with  Alternaria  and  cultural 
tests  confirmed  the  supposition.  The  results  of  these  tests  led  to  the 
conclusion  that  the  fungus  is  prevalent  in  all  lemon-growing  districts 
of  the  state.     While  subsequent  observations  have  confirmed  this,  it 


1  Paper  No.  149,  University  of  California,  Graduate  School  of  Tropical  Agri- 
culture and  Citrus  Experiment  Station,  Eiverside,  California. 

2  Associate  Plant  Pathologist  in  the  Experiment  Station,  Citrus  Experiment 
Station,  Riverside,  California. 

3  Anderson,    H.    W.      Plant    Disease    Survey    Supplement    14:     111.      1921. 
(Reported  by  the  Inspector  for  the  U.  S.  D.  A.  Bureau  of  Markets.) 

4  Fawcett,  H.  S.     The  decay  of  citrus  fruits  on  arrival  and  in   storage  in 
eastern  markets.     Calif.  Citrograph  10:  79,  98-99,  103.     1925. 


4  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

was  observed  also  that  the  fruit  from  certain  districts  or  from  indi- 
vidual packing  houses  showed  more  decay  than  that  from  other  sources. 
This  is  probably  due  to  variations  in  the  vitality  of  the  fruit  or  else 
to  differences  in  the  packing  house  conditions.  It  cannot  be  too 
strongly  emphasized  that  any  storage,  transit  or  market  condition  that 
tends  to  weaken  the  fruit  will  also  tend  to  promote  the  development 
of  Alternaria,  since  this  fungus  usually  develops  very  slowly,  if  at 
all,  in  lemons  of  high  vitality. 

A  systematic  study  of  this  species  of  Alternaria  has  not  been  made. 
However,  its  behavior  in  artificial  cultures  indicates  that  at  least  two 
forms  or  strains,  if  not  species,  may  produce  the  decay  in  question. 
In  some  respects  the  resemblance  to  Altemari  citri,  which  causes  black 
rot  of  the  navel  orange,  is  striking,  but  whether  the  same  organism  is 
involved  in  the  two  cases  has  not  yet  been  determined. 

In  the  eastern  markets  especially,  one  type  of  this  diseased  condi- 
tion of  the  lemon  is  referred  to  as  "center  rot,"  because  of  its  char- 
acteristic invasion  of  the  internal  tissues  before  it  is  evident  on  the 
surface  of  the  fruit.  However,  there  are  other  fungi  which  cause  a 
similar  decay  of  the  lemon  and  for  this  reason  it  seems  best  to  use  here 
the  term  "Alternaria  rot"  rather  than  the  less  specific  term,  "center 
rot." 

ALTERNARIA    ROT   AND    ENDOXEROSIS    COMPARED 

The  terms  Alternaria  rot  and  endoxerosis5  should  not  be  used  inter- 
changeably. Until  1920  the  decay  in  lemon  fruits,  now  known  as 
Alternaria  rot,  was  considered  by  the  growers  and  packers  to  be  the 
advanced  stages  of  endoxerosis.  At  that  time  an  intensive  study  of 
the  disease  was  begun.  Boxes  of  yellow,  silver,  and  green  lemons,  half 
of  each  lot  being  sound  and  the  other  half  affected  with  endoxerosis, 
were  carefully  selected  and  placed  under  the  usual  storage  conditions 
in  each  of  five  packing  houses.  Once  every  month  for  four  months 
a  portion  of  the  fruit  in  each  lot  was  cut  to  detect  any  increase  in 
percentage  of  lemons  showing  endoxerosis  and  any  evidence  of  its 
increase  in  severity  in  those  lemons  affected  when  placed  in  storage. 
Any  of  these  lemons  which  showed  a  more  marked  breaking  down  of 
the  tissues,  either  externally  or  internally,  as  well  as  samples  of  those 
which  had  remained  the  same,  were  brought  to  the  laboratory  where 
cultures  were  made  from  the  tissues. 


s  Endoxerosis  (pronounced  "en-do-zer-6-sis,"  means  internal  drying)  is  a 
technical  term  used  in  place  of  the  less  definite  terms  locally  applied  to  this 
malady,  such  as  "internal  decline,"  "blossom-end  decay,"  "dry  tip,"  and 
"yellow  tip." 


BULL.  408]  ALTERNARIA   ROT   OF   LEMONS  5 

The  observations  in  the  packing*  houses  and  the  results  of  the 
cultural  tests  appeared  to  justify  the  following  conclusions:  (a)  the 
healthy  fruits  did  not  become  affected  with  endoxerosis,  (6)  those 
already  affected  with  endoxerosis  did  not  show  any  increase  of  the 
characteristic  symptoms,  (c)  in  the  cultures  no  fungus  could  be 
isolated  from  the  healthy  fruits  or  from  those  having  only  the 
symptoms  of  endoxerosis  as  it  appears  in  the  fruits  taken  directly 
from  the  trees,  and  (d)  the  cultures  showed  that  actual  decay  of  the 
healthy  lemons  and  of  those  having  endoxerosis  was  caused  in  every 
case  by  a  fungus.  In  almost  every  case  Alternaria  was  found  to  be 
the  cause  of  the  decay  except,  of  course,  where  molds  had  gained 
entrance  through  mechanical  injuries  due  to  handling.  In  only  a  few 
isolated  cases  were  such  fungi  as  Colletotrichum  and  Phomopsis  found 
to  be  the  principal  causes  of  the  decay. 

In  1921  a  similar  experiment  was  conducted  in  collaboration  with 
15  lemon  association  packing  houses.  The  houses  were  widely  distri- 
buted over  the  lemon-growing  districts  of  southern  California.  After 
the  experiment  was  started  the  individual  tests  were  put  into  the  care 
of  the  manager  or  foreman,  or  both,  of  the  respective  packing  houses 
with  the  instruction  (a)  that  they  make  the  observations,  (&)  that 
they  send  fruits  to  the  laboratory  for  making  cultures,  and  (c)  that 
upon  the  termination  of  the  test  they  report  their  conclusions. 

The  results  of  the  cultural  tests  were  identical  with  those  made  in 
1920.  The  breaking  down  of  the  tissues  of  those  fruits  affected  with 
endoxerosis  did  not  progress,  unless  they  were  infected  by  Alternaria. 
Reports  from  thirteen  of  the  packing  house  managers  or  foremen 
showed  that  no  sound  lemons  became  affected  with  endoxerosis  after 
they  were  placed  in  storage.  Of  the  other  two,  one  reported  incon- 
clusive results  and  one  reported  that  "The  decline  (endoxerosis)  con- 
tinued to  advance  in  the  affected  fruit  resulting  in  a  final  complete 
drying-up  or  slushy  rot."  This  last  statement  does  not  in  any  way 
contradict  the  conclusion  that  endoxerosis  does  not  progress  in  the 
fruit  after  it  is  taken  from  the  tree  because  the  symptoms  mentioned 
are  those  of  Alternaria  and  not  of  endoxerosis.  The  statement  serves 
to  indicate  the  confusion  previously  existing  as  to  the  identity  of  the 
two  diseases.  To  prevent  future  confusion  it  may  be  well  at  this  time 
to  state  the  characteristic  symptoms  of  these  two  maladies.  Plate  1 
illustrates  many  of  the  characteristic  differences  between  these  two 
diseases.6 


6  See  also  Fawcett,  H.  S.,  and  H.  A.,  Lee.  Citrus  diseases.  McGraw-Hill 
Book  Co.,  N.  Y.  (For  Alternaria  rot  see  fig.  130,  A  to  D,  for  endoxerosis  see 
fig.  149.) 


UNIVERSITY    OF    CALIFORNIA— EXPERIMENT    STATION 


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Bull.  408] 


ALTERNARIA    ROT    OF    LEMONS 


The  results  of  these  two  sets  of  experiments,  performed  in  1920  and 
in  1921,  together  with  widespread  observations  since  that  time,  have 
shown  conclusively  that  the  breakdown  and  decay  of  the  fruits  in 
question  is  due  to  the  action  of  the  fungus  Alternaria  and  not  to  the 
so-called  " later  stages"  of  endoxerosis. 


INOCULATION    EXPERIMENTS 

During  the  course  of  the  preceding  observations  and  experiments 
the  question  arose  as  to  whether  the  lemon  fruits  became  infected  with 
Alternaria  before  or  after  being  picked.  An  effort  was  made  to 
answer  this  question  by  making  inoculation  experiments  first  in  two 
packing  houses  and  later  under  controlled  conditions  in  the  laboratory. 

TABLE  1 

Inoculation  Experiments  in  the  Packing  House.  Comparative  Percentages 
of  Alternaria  Rot  Appearing  in  Inoculated  and  Uninoculated  Sound  and 
Endoxerotic  Lemons  after  90  Days  in  Storage. 


Proportion 

of  lemons 

affected  with 

endoxerosis 

Lemons  infected  by  Alternaria 

Average 

Condition  of  lemons 

Inoculated 

Checks 

percentage  of 
Alternaria 

Yellow,  sound 

Per  cent 

20 
75 
15 
50 
10 
15 

Per  cent 

17 
35 

9 
20 

5 
10 

Per  cent 

6 
25 

9 
30 

3 

6 

Per  cent 
12 

Yellow,  endoxerotic... 

30  • 

Silver,  sound 

9 

Silver,  endoxerotic 

Green,  sound 

Green,  endoxerotic 

25 

4 

8 

In  the  packing  houses. — Two  hundred  lemons  of  each  of  the  kinds 
indicated  in  table  1  were  taken  from  the  washer  and  dried.  Before 
these  fruits  were  placed  under  the  usual  storage  conditions,  those 
designated  as  "inoculated"  were  "painted"  with  a  suspension  of 
Alternaria  spores,  usually  on  the  stem  end  around  the  button  but  in 
some  cases  on  both  stem  and  stylar  ends.  At  the  end  of  each  of  three 
30-day  intervals  the  percentages  of  Alternaria  appearing  in  each  lot 
of  fruit  were  determined  (table  1).  The  fruit  in  one  packing  house 
showed  a  slightly  higher  percentage  of  Alternaria  in  each  lot  than  that 
in  the  other  house,  but  the  ratios  between  the  different  kinds  of  fruit 
were  similar. 

The  results  are  not  conclusive.  They  show  a  higher  percentage  of 
Alternaria  rot  in  the  inoculated  yellows  and  greens  but  not  in  the 
silvers.    While  the  results  show  that  in  four  of  the  six  lots  the  amount 


8  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

of  Alternaria  rot  appearing  within  this  three-month  period  was 
apparently  increased  through  inoculation,  yet  they  clearly  do  not 
prove  that  none  of  the  lemons  in  any  of  the  lots  would  have  decayed 
without  inoculation.  The  results  of  some  of  the  experiments  to  be 
described  a  little  farther  on  in  this  paper  will  give  additional  evi- 
dence on  this  question.  No  infections  resulted  from  the  stylar-end 
inoculations. 

A  point  of  particular  interest  in  connection  with  this  experiment  is 
the  fact  that  a  much  larger  percentage  of  Alternaria  rot  appeared  in 
the  endoxerotic  than  in  the  sound  lemons,  as  indicated  in  the  last 
column  of  table  1.  These  and  other  experiments  and  observations 
indicate  that  this  was  due  to  the  lower  vitality  of  endoxerotic  lemons, 
which  makes  them  less  able  to  resist  invasion  by  the  fungus. 

In  the  laboratory. — The  inoculation  experiments  in  the  laboratory 
were  confined  to  silver  and  light-green  lemons.  They  were  carefully 
sorted  and  only  those  of  the  best  quality  were  used.  Each  group  of 
silvers  and  light  greens  was  divided  into  twenty-one  lots  of  twenty 
lemons  each.  Of  the  twenty-one  lots  of  silvers  seven  were  used  as 
controls,  seven  were  soaked  five  minutes  in  copper  sulphate  solution 
(%o  of  1  per  cent,  as  commonly  used  in  the  lemon  washer  in  the 
packing  houses)  and  the  other  seven,  after  soaking  as  in  the  preceding 
case,  were  dried  and  then  inoculated  with  a  suspension  of  a  pure 
culture  of  Alternaria  spores  in  water.  The  suspension  was  applied  by 
means  of  a  camel's-hair  brush  and,  in  so  far  as  possible,  was  applied 
under  as  well  as  around  and  on  top  of  the  buttons.  The  twenty-one 
lots  of  light  greens  were  divided  and  treated  in  the  same  manner.  All 
lemons  were  wrapped  in  standard  wrappers.  The  lots  of  silvers  and 
light  greens  were  then  divided  into  seven  groups  in  such  a  manner 
that  each  group  contained  one  lot  of  silvers  for  controls,  one  that  had 
been  soaked,  one  that  had  been  soaked  and  then  inoculated  and  three 
similar  lots  of  the  light  greens.  Each  group  therefore  included  sixty 
silvers  and  sixty  light  greens.  One  of  these  seven  groups  was  then 
placed  in  each  of  the  seven  compartments  of  a  constant  temperature 
apparatus.  The  respective  temperatures  of  these  compartments  were 
approximately  48°,  59°,  68°,  75°,  82°,  90°  and  97°  F.  The  tempera- 
ture in  any  one  compartment  did  not  vary  more  than  one  or  two 
degrees  during  the  course  of  the  experiment.  The  relative  humidity 
in  each  compartment  was  at  or  near  the  saturation  point.  At  intervals 
of  from  one  to  three  weeks  for  a  period  of  three  and  a  half  months  the 
fruit  was  examined  and  the  number  in  each  lot  that  showed  visible 
signs  of  decay  was  recorded  and  cultures  made,  when  necessary,  to 
identify  the  fungus  causing  the  decay.  A  summary  of  the  results  of 
this  experiment  is  shown  in  table  2. 


Bull.  408] 


ALTERNARIA    ROT    OF    LEMONS 


TABLE  2 

Effect  of  Attempted  Sterilization  and  of  Artificial  Inoculation  on  the 
Amount  of  Alternaria  Rot  Appearing  in  Sound  Silver  and  Light-green 
Lemons,  under  Controlled  Temperature  Conditions. 


Kind  of  lemons 

Treatment 

Per  cent  Alternaria  rot  at  the  following 
temperatures,  °F 

Average 
percentage 

48 

59 

68 

75 

82 

90 

97 

Alternaria 
rot 

Silvers 

Checks 

Soaked  in  CuS04... 
|  Soaked  in  C11SO4, 
inoculated 

0 

10 

15 

0 

10 

20 

5 
25 

15 

10 
20 

30 

40 
40 

40 

50 
35 

50 

20 
15 

20 

18 
22 

27 

Greens 

Checks 

J  Soaked  in  CUSO4.... 
Soaked  in  CuS04, 
inoculated 

0 
0 

0 

5 
0 

5 

0 
5 

0 

10 
15 

5 

45 
35 

55 

25 
20 

35 

25 
25 

35 

16 
14 

19 

The  results  of  this  experiment  show  that : 

1.  The  copper  sulphate  solution  had  no  sterilizing  effect  on  the 
lemons,  in  so  far  as  preventing  development  of  Alternaria  rot  was 
concerned. 

2.  Artificial  inoculation  of  the  lemons  apparently  increased  to  a 
limited  extent  the  percentage  of  Alternaria  rot  appearing  in  the 
lemons,  within  the  time  limits  of  the  experiment,  as  was  also  shown 
in  table  1.  However,  the  data  in  table  3  should  be  taken  into  con- 
sideration in  this  connection.  It  may  be  said  also  that  the  water 
added  at  the  time  of  making  the  inoculation  was  probably  an  important 
factor  in  increasing  the  amount  of  decay  in  the  inoculated  lemons; 
the  added  water  increased  the  amount  of  moisture  under  the  buttons 
and  thus  made  conditions  more  favorable  for  the  growth  of  the 
Alternaria. 

3.  The  optimum  temperature  for  the  development  of  Alternaria  in 
these  lemons  proved  to  be  in  the  neighborhood  of  82°  F.  Subsequent 
experiments  and  observations  have  shown  that  this  is  approximately 
the  optimum  temperature  for  the  development  of  Alternaria  rot  in 
lemons  in  general. 

On  the  date  of  the  last  examination  all  of  the  lemons  were  removed 
from  the  constant-temperature  apparatus.  A  large  number  of  them 
still  showed  no  exterior  indications  of  decay.  This  was  especially 
true  of  those  that  had  been  kept  at  the  lower  temperatures.  As  the 
lemons  were  removed  from  the  compartments  each  was  cut  transversely 
just  below  the  button  to  detect  any  case  of  Alternaria  rot  in  the  early 


10 


UNIVERSITY    OP    CALIFORNIA EXPERIMENT    STATION 


stages  of  development.  The  percentage  in  each  compartment  showing 
the  development  of  Alternaria  is  given  in  table  3.  The  extent  of 
invasion  of  the  Alternaria,  from  the  buttons  into  the  central  tissues 
of  the  lemons,  ranged  from  about  an  eighth  to  three-quarters  of 
an  inch. 

TABLE  3 

Percentages  of  the  Externally  Sound  Lemons  of  Table  2,  which,  when 
Removed  from  the  Compartments  of  the  Constant -Temperature  Appa- 
ratus and  Cut  Across  just  Below  the  Buttons,  Showed  that  Alternaria 
had  Begun  to  Invade  the  Tissues. 


Per  cent  silvers  having 
Alternaria  rot 

Average 

Per  cent  greens  having 
Alternaria  rot 

Temperature 
°F 

Controls 

Soaked 
in  CuS04 

Soaked 
in  CuSO*, 
inoc- 
ulated 

Controls 

Soaked 
in  CuS04 

Soaked 
inCuSO*. 
inoc- 
ulated 

Average 

97 

100 

100 

100 

100 

100 

100 

100 

100 

90 

100 

100 

100 

100 

98 

96 

99 

98 

82 

100 

98 

99 

99 

91 

89 

88 

89 

75 

100 

91 

92 

94 

78 

75 

78 

77 

68 

79 

69- 

76 

75 

70 

79 

60 

70 

59 

55 

72 

69 

65 

42 

45 

42 

43 

48 

55 

61 

59 

58 

35 

45 

40 

40 

Average.... 

84 

84 

85 

84 

73 

76 

72 

74 

The  data  in  table  3  show : 

1.  A  large  percentage  of  the  apparently  sound  lemons  was  found 
to  be  already  infected  with  Alternaria  in  the  early  stages.  This  was 
especially  true  of  the  higher  temperatures.  The  fact  that  at  the  tem- 
peratures of  97°  and  90°  F  a  larger  percentage  of  the  lemons  showed 
infection  than  at  82°  F,  which  has  been  given  as  the  approximate 
optimum  for  the  development  of  Alternaria.  in  lemons,  might  be  taken 
to  indicate  that  82°  F  is  really  not  the  optimum.  However,  the  slightly 
greater  percentage  of  infection  at  the  highest  temperatures,  which 
are  above  the  optimum  for  the  growth  of  the  fungus,  may  have  been 
due  to  a  weakened  condition  of  the  lemon  tissues  resulting  from 
exposure  to  these  high  temperatures.  Examination  showed  that  the 
fungus  had  developed  much  farther  in  the  lemons  held  at  82°  F  than 
in  those  held  at  the  higher  temperatures. 

2.  Averaging  the  data  for  all  temperatures,  the  uninoculated  silvers 
had  only  one  per  cent  less  infection  than  the  inoculated,  while  in  the 
light  greens  the  two  groups  of  uninoculated  had  one  and  four  per  cent 


Bull.  408] 


ALTERNARIA    ROT    OF    LEMONS 


11 


respectively  more  infection  than  the  inoculated.  This  indicates  that 
the  Alternaria  organism  was  already  present  in  or  beneath  each  lemon 
button  when  the  lemons  were  picked  and  with  sufficient  time  and 
favorable  conditions  practically  all  of  these  lemons  would  have  shown 
decay  without  artificial  inoculation. 

3.  The  infection  was  approximately  ten  per  cent  greater  in  the 
silvers  than  in  the  light  greens,  showing,  as  was  indicated  in  tables  1 
and  2,  that  the  longer  the  lemons  are  left  on  the  trees  after  becoming 
practically  mature,  the  sooner  will  they  show  decay  by  Alternaria 
after  being  placed  in  storage.  The  data  in  table  5  emphasize  the 
correctness  of  this  statement  even  more  markedly. 


GROWTH    RATE    OF    ALTERNARIA    MYCELIUM 

Having  found  that  82°  F  was  approximately  the  optimum  tem- 
perature for  the  development  of  Alternaria  rot  in  the  lemons,  it  seemed 
advisable  to  determine  next  the  optimum  temperature  for  the  rate  of 
growth  of  Alternaria  in  artificial  cultures.  For  this  purpose  the  same 
constant-temperature  apparatus  was  used  but,  as  indicated  in  table  4, 

TABLE  4 

The  Effect  of  Different  Temperatures  on  the  Rate  of  Growth  of  Alter- 
naria Mycelium  on  an  Artificial  Culture  Medium  (Cornmeal  Agar). 
Transfers  were  Made  on  January  6,  at  9  A.M. 


Diameters  of  mycelial  discs  growing  at  the  following  temperatures,  °F 

Times  of  measurement 

48 

58 

66 

72 

79 

85 

94 

Jan.  8,  9  a.m 

Jan.  9,  9  a.m 

Jan.  10,  9  a.m 

cm 

0.4 
0.5 
0.9 

cm 

0.8 
1.7 
1.9 

cm 

1.6 
2.3 
3.0 

cm 

1.6 

2.8 
3.6 

cm 

2.1 
3.3 
5.2 

cm 

2.0 
3.0 
4.2 

cm 

0.7 
0.9 
1.2 

the  range  of  temperatures  was  a,  little  lower  than  in  the  preceding 
experiment.  Sterilized  Petri  dishes  and  glucose-potato  and  cornmeal 
agar  were  used.  A  very  small  droplet  of  water  containing  Alternaria 
spores  in  suspension  was  placed  on  the  center  of  the  culture  medium 
in  each  Petri  dish  by  means  of  a  small,  sterilized  platinum  loop.  An 
equal  number  of  dishes  of  the  two  kinds  of  culture  media  was  then 
placed  in  each  of  the  different  constant-temperature  compartments. 
It  is  characteristic  of  Alternaria  mycelium,  when  grown  on  culture, 
media  of  this  kind,  to  grow  approximately  equally  in  a  plane  in  all 
directions  from  the  original  point  of  inoculation,  thus  making  a  disc- 
shaped mat.     The  rate  of  growth  was  determined  by  measuring  at 


12  UNIVERSITY   OF    CALIFORNIA — EXPERIMENT    STATION 

intervals  the  diameter  of  each  disc  of  mycelium.  While  the  character 
of  the  mycelium  on  the  glucose-potato  agar  was  a  little  different  from 
that  on  cornmeal  agar,  the  rate  of  growth  on  the  two  was  so  nearly 
the  same  that  only  the  averages  for  the  latter  are  given  (table  4). 

The  data  show  (a)  that  the  optimum  temperature  for  the  growth  of 
Alternaria  on  these  two  artificial  culture  media  is  approximately  the 
same  as  in  lemon  fruits  (82°  F),  and  (&)  that  Alternaria  will  grow 
slowly  at  least,  at  temperatures  as  low  as  48°  and  as  high  as  94°  F. 
These  results  agree  very  well  with  the  data  obtained  on  the  develop- 
ment of  Alternaria  in  lemon  tissues  (table  3). 


RESISTANCE  OF  ISOLATED  ALTERNARIA  SPORES  AND   MYCELIUM 

Tests  on  the  resistance  of  Alternaria  spores  and  mycelium  to 
mercuric  chloride  and  to  hot  water  were  made,  preliminary  to  test- 
ing the  efficiency  of  various  sterilizing  solutions  for  the  prevention 
of  Alternaria  rot  in  lemons. 

Mercuric  chloride, — Pure  cultures  of  Alternaria,  isolated  from 
lemons,  were  grown  on  nutrient  agar  in  Petri  dishes.  As  soon  as  the 
surface  of  the  medium  became  well  covered  with  mycelium  and  spores, 
small  pieces  of  the  agar  (approximately  %e  X  Vie  X  %  inch)  were 
removed  and  placed  in  small  glass  funnels  lined  with  filter  paper. 
The  funnels  were  then  immersed  in  a  solution  of  1  part  mercuric 
chloride  to  1000  parts  of  water  and  kept  at  a  temperature  of  75°  F. 
After  periods  of  2,  4,  6,  8,  and  10  minutes  respectively  a  given  number 
of  the  funnels  was  removed  from  the  sterilizing  solution  and  washed 
repeatedly  with  sterile  distilled  water  for  half  an  hour  to  remove  all 
traces  of  mercuric  chloride.  The  pieces  of  agar,  bearing  the  spores 
and  mycelium,  were  then  transferred  to  fresh  culture  medium  in  Petri 
dishes  and  incubated  at  77°  F.  Small  pieces  of  the  original  culture 
which  had  not  been  sterilized  were  washed  with  sterile  distilled  water 
and  similarly  incubated  to  serve  as  checks. 

All  of  the  unsterilized  cultures  showed  the  characteristic  develop- 
ment of  Alternaria  while  in  all  of  the  treated  cultures  growth  was 
entirely  lacking. 

The  results  of  this  experiment  show  that  Alternaria  spores  and 
mycelium,  grown  on  artificial  culture  media,  may  be  killed  by  immers- 
ing them  for  only  two  minutes  in  a  solution  of  1  to  1000  mercuric 
chloride  and  water,  kept  at  75°  F. 

In  another  experiment  mature  fruits  and  twigs  were  brought  to 
the  laboratory  and  their  surfaces  washed  with  a  brush  dipped  in  sterile 
distilled  water.     Portions  of  the  water  were  then  filtered  through 


BULL.  408]  ALTERNARIA   ROT   OP   LEMONS  13 

filter  paper  in  the  small  funnels  used  in  the  previous  experiment. 
Microscopical  examination  showed  the  presence  of  an  abundance  of 
Alternaria  spores  and  some  bits  of  mycelium.  The  mycelium,  where 
present,  was  composed  of  small,  irregular  pieces  such  as  one  would 
expect  to  find  produced  under  adverse  conditions.  The  process  of 
sterilization  was  the  same  as  in  the  preceding  experiment.  After 
washing,  small  masses  of  the  spores  and  mycelium  were  scraped  from 
the  filter  papers,  transferred  to  culture  dishes  and  incubated  at  77°  F. 

The  results  were  similar  to  those  of  the  preceding  experiment.  The 
unsterilized  checks  showed  100  per  cent  growth  but  there  was  no 
growth  in  any  of  the  cultures  where  the  spores  and  mycelium  had 
been  exposed  to  the  sterilizing  solution  for  periods  of  2,  4,  6,  8,  or  10 
minutes.  Evidently  the  Alternaria  organism  whether  obtained  from 
artificial  or  natural  cultures  may  be  killed  by  immersion  for  a  period 
of  only  two  minutes  in  a  solution  of  1  part  mercuric  chloride  in  1000 
parts  of  water  at  a  temperature  of  75°  F. 

Hot  water. — Since  hot  water  at  a  temperature  of  115  to  125°  F 
is  commonly  used  in  the  lemon  packing  houses  as  a  means  of  controlling 
brown  rot  (Pythiacystis  citrophthora) ,  it  seemed  desirable  to  deter- 
mine the  effects  of  hot-water  treatment  on  Alternaria,  This  test  was 
conducted  in  the  same  manner  as  the  two  preceding  ones,  except  that 
hot  water  (116°  F)  was  used  as  the  sterilizing  medium  instead  of 
mercuric  chloride. 

In  this  test  all  cultures  showed  100  per  cent  Alternaria  infection. 
It  was  interesting  to  note,  however,  that  the  treatment  retarded  the 
rate  of  growth,  especially  in  the  cultures  that  had  been  exposed  for 
ten  minutes. 


RESISTANCE    OF    ALTERNARIA    SPORES    AND     MYCELIUM     WHILE 

ADHERING    TO    THE    SURFACE    OF    LEMON 

FRUITS   AND   TWIGS 

The  mercuric  chloride  solution  was  tested  next  on  spores  and 
mycelium  in  contact  with  the  surface  of  the  tissues.  Thin  strips  of 
peel  taken  from  mature  lemon  fruits  were  first  used.  Microscopical 
study  showed  that  the  mycelium  had  not  penetrated  the  surface  layer 
of  cells.  The  strips,  approximately  %  inch  thick,  were  cut  into  pieces 
about  %  inch  square.  Forty  of  these  pieces  were  put  into  each  of 
five  cheese-cloth  bags,  and  vacuumized  in  a  flask  of  water  for  one  hour 
to  remove  all  air  that  might  prevent  the  sterilizing  solution  from 
coming  into  contact  with  the  spores  or  mycelium.  Upon  removal  from 
the  vacuum  flask  four  of  the  bags  were  immersed  in  the  sterilizing 


14  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

solution.  They  were  removed  successively  from  this  solution  at 
intervals  of  2,  4,  6,  and  8  minutes.  After  thorough  washing  to  remove 
the  mercuric  chloride,  the  pieces  of  peel  were  plated  on  a  culture 
medium  and  incubated  at  77°  P.  The  pieces  of  peel  from  the  fifth 
bag  were  not  sterilized  but  were  cultured  in  the  same  way  as  the  others, 
to  serve  as  checks.  The  sterilizing  solution  was  the  same  as  before 
and  was  used  at  73°  P. 

The  percentages  of  Alternaria  appearing  in  these  cultures  at  the 
end  of  eight  days  were  as  follows:  checks,  35  per  cent;  immersed 
2  minutes,  18  per  cent ;  4  minutes,  13  per  cent ;  6  minutes,  8  per  cent ; 
and  8  minutes,  none. 

The  second  part  of  the  test  was  similar  to  the  first  part  except  that 
portions  of  twigs  were  used  instead  of  pieces  of  peel,,  and  they  were 
immersed  in  the  sterilizing  solution  for  only  two  minutes.  The 
portion  of  the  twig  used  was  about  %  inch  of  the  pedicel,  the 
portion  of  the  stem  just  below  the  button.  This  part  of  the  twig  is 
free  from  bracts,  which  might  cover  the  spores,  and  to  the  unaided  eye 
it  appears  to  be  entirely  smooth.  Sixty-four  of  these  pieces  were 
treated  and  then  cultured  as  in  the  first  part  of  the  test. 

As  in  the  case  of  the  peel  it  was  found  that  a  two-minute  exposure 
to  the  sterilizing  solution  was  not  sufficient  to  kill  all  of  the  Alternaria, 
After  one  week  in  the  incubator  19  per  cent  of  the  pieces  showed  a 
growth  of  Alternaria. 

The  results  of  both  parts  of  the  preceding  test  indicate  rather 
strongly  that  even  though  an  exposure  of  isolated  spores  or  mycelium 
to  a  sterilizing  solution  for  a  given  period  will  kill  them  it  is  not  safe 
to  conclude  that  a  like  exposure  will  kill  them  when  they  are  in 
contact  with,  but  not  embedded  in,  the  tissues  which  at  least  in  some 
cases  they  may  enter  later.  Had  not  previous  examination  shown  that 
such  was  not  the  case  one  might  have  concluded  that  the  reason  for 
the  growth  of  Alternaria  in  connection  with  some  of  the  pieces  of  peel 
or  twig  was  because  the  mycelium  had  already  penetrated  the  tissues 
and  was  out  of  reach  of  the  sterilizing  solution. 

STERILIZATION   OF  LEMON   FRUITS  AS   A   MEANS  OF   PREVENTING 

ALTERNARIA    ROT 

The  next  step  in  the  study  was  an  attempt  to  at  least  minimize  if 
not  entirely  prevent  this  decay  by  sterilizing  the  fruits.  The  plan 
followed  was  (1)  to  use  mercuric  chloride  in  the  wash  water  in  the 
packing  house,    (2)    to   use   "brogdite"7  in  the  wash  water  in  the 


7  Brogdite  is  a  commercial  sterilizing  medium  that  is  being  used  in   some 
packing  houses  to  prevent  decay  caused  by  blue  and  green  molds. 


Bull.  408]  ALTERNARIA  rot  of  lemons  15 

packing  house,  (3)  to  remove  the  buttons,  and  (4)  to  test  the  efficiency 
of  various  sterilizing  solutions  in  the  laboratory,  under  controlled 
conditions,  provided  the  other  methods  did  not  prove  to  be  effective 
or  practicable. 

1.  Tests  were  made  in  two  packing  houses.  In  one  house  five  boxes 
of  silver  lemons  and  twenty-two  boxes  of  green  lemons  were  washed 
in  each  of  the  following:  (a)  water  at  approximately  95°  F,  (b) 
mercuric  chloride  sterilizing  solution  at  60°  F,  and  (c)  the  same 
solution  at  110°  F.  The  lemons  were  retained  in  the  solution  approxi- 
mately five  minutes,  then  rinsed  in  the  usual  manner  and  put  into 
storage. 

The  fruit  in  the  other  packing  house  (three  boxes  of  yellows  and 
five  boxes  each  of  silvers  and  greens)  was  treated  in  the  same  manner. 
The  fruit  in  both  houses  was  examined  at  intervals  for  three  and  a 
half  months. 

The  results  of  the  tests  in  these  two  packing  houses  indicate  that 
mercuric  chloride  can  not  be  considered  an  efficient  sterilizing  medium 
for  the  prevention  of  Alternaria  rot  in  lemons.  Apparently  the  cold 
mercuric  chloride  solution  had  no  controlling  effect  whatsoever,  and 
while  the  results  with  the  hot  solution  indicated  a  slight  degree  of 
control  it  was  considered  to  be  more  of  a  retarding  than  an  actual 
sterilizing  effect. 

2.  In  the  test  with  brogdite  eight  boxes  of  yellow  lemons  (1700 
fruits)  were  divided  into  four  lots  of  two  boxes  each.  The  first  lot 
was  washed  in  water  at  approximately  113°  F  and  the  second,  third, 
and  fourth  lots  were  immersed  in  brogdite  solution  of  the  same  tem- 
perature for  5,  5,  and  10  minutes  respectively,  after  which  they  were 
rinsed  in  cold  water.  The  strength  of  the  solution  for  the  second  lot 
was  sixteen  ounces  of  brogdite  to  a  gallon  of  water,  and  for  the  third 
and  fourth  lots,  twenty-four  ounces. 

By  the  end  of  two  months  2,  4,  16,  and  8  per  cent  respectively  of 
Alternaria  rot  had  appeared  in  the  four  lots.  AVhile  only  one  test 
was  made  with  this  solution  at  this  time  the  results  indicated  that  it 
would  be  useless  to  repeat  it.  That  more  Alternaria  rot  appeared  in 
the  treated  than  in  the  untreated  lemons  indicates  that  the  treatment 
produced  or  hastened  some  change  in  the  lemon  tissues  which  permitted 
a  more  rapid  invasion  by  the  fungus.  It  should  be  said,  however, 
that  there  was  no  external  indication  of  injury  due  to  the  treatment. 

3.  In  each  of  four  packing  houses,  six  boxes  of  yellow  lemons8  and 
six  boxes  of  green  lemons  were  given  the  usual  treatment  of  washing 

8  Although  these  lemons  were  yellow  they  were  not  classed  as  "tree-ripes. " 
They  had  been  prematurely  colored  by  the  winds  and  comparatively  low  tempera- 
tures prevailing  during  November  and  December. 


16 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


and  sorting.  Three  boxes  of  yellows  and  three  of  greens  were  placed 
under  storage  conditions.  The  remaining  three  boxes  each  of  yellows 
and  greens  were  sweated  to  such  an  extent  as  to  cause  at  least  most 
of  the  buttons  to  drop.  This  was  done  to  determine  whether  the 
removal  of  the  buttons  would  cause  a  reduction  of  the  amount  of  Alter- 
naria  rot.  In  two  of  the  houses  the  sweating  was  done  with  ethylene 
gas  and  in  the  other  two  with  oil  stoves,  The  sweating  periods  in 
packing  houses  1  to  4  were  8,  8,  7,  and  8  days  respectively.  The  sweat- 
ing caused  no  visible  injury  to  the  fruit.  At  the  end  of  the  sweating 
period  any  buttons  still  attached  were  removed  by  hand.  These  boxes 
of  lemons  were  then  placed  in  the  storeroom  beside  the  untreated 
checks.  All  boxes  of  lemons  were  examined  at  intervals  for  four 
months  after  the  last  boxes  were  placed  in  storage.  The  total  number 
of  lemons  in  each  lot  showing  Alternaria  rot  within  that  period  is 
shown  in  table  5.  The  exact  number  of  lemons  used  was  not  deter- 
mined but  there  were  from  375  to  400  in  each  box. 

TABLE  5 

The  Amount  of  Alternaria  Rot  in  Lemons  as  Influenced  by  the  Removal 
of  the  Buttons.     Three  Boxes  in  Each  Lot. 


Sweated,  buttons  removed 

Checks,  buttons  not  removed 

Packing  house 

Yellow 

Green 

Yellow 

Green 

Number  of  lemons  showing  Alternaria  rot 

1 

157 
55 
20 
46 

1 

13 

3 

49 

223 
30 
21 

118 

2 

2 

3 

3 

2 

4 

2 

The  results  indicate  that  the  removal  of  the  buttons  from  the 
lemons  is  not  a  feasible  method  for  the  control  of  Alternaria  rot.  By 
referring  to  the  table  it  may  be  seen  that  in  packing  house  No.  1  there 
was  apparently  30  per  cent  control  and  in  house  No.  4,  20  per  cent,  but 
in  house  No.  3  there  was  no  indication  of  control,  and  in  house  No.  2 
there  was  51  per  cent  more  Alternaria  rot  in  those  lemons  which  had 
the  buttons  removed  than  in  the  checks, 

A  similar  test  was  later  conducted  in  the  laboratory.  In  this  case 
all  of  the  lemons  were  silvers  and  the  buttons  were  removed  by  hand 
rather  than  by  sweating.  The  fruit  was  brought  directly  from  the 
grove  and  stored  in  field  boxes  in  a  basement  room  of  the  laboratory 
where  the  temperature  did  not  vary  more  than  four  degrees,  remaining 


BULL.  408]  ALTERNARIA   ROT   OF   LEMONS  17 

for  the  entire  time  of  the  test  (December  21  to  March  5)  at  approxi- 
mately 70°  F.  This  temperature,  although  below  the  optimum,  is  very 
favorable  for  the  growth  of  Alternaria.  The  humidity  of  the  room  was 
such  that  by  the  end  of  the  test  the  lemons  showed  about  the  same 
amount  of  shrinkage  as  would  have  occurred  under  the  usual  storage 
conditions  in  the  packing  house. 

On  January  8  none  of  the  fruits  showed  decay,  but  the  tempera- 
ture and  humidity  had  been  favorable  for  the  germination  of  Alter- 
naria spores  that  were  attached  to  the  buttons  and  to  the  surface  of 
the  fruits.  Small  mats  of  mycelium  could  be  distinguished  very  easily 
with  the  unaided  eye.  The  appearance  of  these  mats  of  mycelium, 
as  they  formed  on  the  buttons  in  this  and  other  tests  and  as  they  often 
occur  under  packing  house  conditions,  is  shown  in  plate  2,  figures  A 
and  B.  It  was  especially  interesting  that  more  mats  of  mycelium  could 
be  seen  in  the  button  pits  of  those  lemons  from  which  the  buttons  had 
been  removed  than  could  be  seen  in  connection  with  the  buttons  that 
remained  attached  to  the  lemons.  This  condition  gives  further  evi- 
dence that  removal  of  the  buttons  does  not  necessarily  remove  all 
chances  of  infection.  Data  to  be  presented  farther  on  will  show  that, 
although  the  button  be  removed,  there  are  left  in  the  crevices  in  the 
tissues  under  the  button  a  sufficient  number  of  spores  and  bits  of 
resistant  mycelium  to  cause  infection  as  soon  as  conditions  are 
favorable. 

By  March  5,  sixty -three  cases  of  Alternaria  rot  had  been  found  in 
those  lemons  from  which  the  buttons  had  been  removed,  and  only  six 
in  the  checks,  to  which  the  buttons  remained  attached.  These  results 
are  comparable  to  those  in  packing  house  No.  2,  as  shown  in  table  5.  It 
may  be  of  interest  to  note  here  that  although  the  buttons  in  this  test 
were  forcibly  removed  by  using  a  clipper,  such  as  is  used  in  picking 
lemons,  only  eight  lemons  in  the  entire  lot  showed  a  growth  of 
Penicillium. 

Some  of  the  reasons  why  removal  of  the  buttons  may  favor  infection 
by  Alternaria  are  (a)  the  removal  of  the  buttons  may  weaken  the 
tissues,  thus  making  them  more  susceptible  to  the  attack  of  the  fungus, 
(b)  the  evaporation  from  the  exposed  tissues  may  make  conditions 
more  favorable  for  the  growth  of  the  fungus,  and  (c)  this  excessive 
evaporation  may  produce  changes  in  the  lemons  which  make  them 
more  susceptible  to  attack. 

Although  only  six  lemons  in  the  checks  showed  external  signs  of 
Alternaria  rot  further  examination  disclosed  the  fact  that  a  large 
percentage  of  them  were  in  the  early  stages  of  infection  and  would 
soon  have  become  decayed.     This  was  determined  by  taking  thirty 


18  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

lemons  at  random  from  among  the  checks.  A  thin  slice  was  cut  from 
the  stem  end  of  each  lemon  in  a  manner  such  that  the  knife  passed 
just  below  the  juncture  of  fruit  and  button,  thus  removing  all  crevices 
which  might  be  harboring  spores  or  mycelium.  A  small  disc  of  tissues 
was  then  taken  from  each  lemon  immediately  below  the  original  point 
of  attachment  of  the  button,  and  cultured  to  determine  the  number 
that  were  infected.  Twenty-one  (70  per  cent)  of  the  thirty  discs 
showed  an  abundant  growth  of  Alternaria. 

The  attempt  to  control  Alternaria  rot  was  continued  by  immersing 
lemons  in  sterilizing  solutions  after  the  buttons  had  been  removed. 
In  the  first  part  of  the  test  silver  lemons  that  had  just  been  picked 
and  washed  were  brought  from  three  packing  houses.  The  buttons 
were  removed  from  half  of  each  lot  and  both  lots  were  immersed  for 
five  minutes  in  mercuric  chloride  of  the  usual  strength,  at  65°  F. 
They  were  then  put  into  glass  containers  and  placed  on  tables  in  the 
laboratory  where  the  temperature  ranged  from  54°  to  72°  F.  Observa- 
tions were  made  at  intervals  for  a  period  of  five  months.  The  com- 
parative amounts  of  Alternaria  rot  appearing  in  the  different  lots  dur- 
ing this  time  were :  from  house  No.  1,  8  per  cent  in  those  without 
buttons  and  17  per  cent  in  checks ;  house  No.  2,  67  per  cent  in  those 
without  buttons  and  68  per  cent  in  checks;  and  from  house  No.  3, 
8  per  cent  in  those  without  buttons  and  67  per  cent  in  checks. 

The  second  part  of  the  test  was  like  the  first  part  except  that  only 
one  lot  of  lemons  was  used  and  the  sterilizing  solution  was  copper 
sulphate  (%0  °f  1  Per  cent>  as  commonly  used  in  lemon  packing 
houses).  The  results  of  this  part  of  the  test  showed  51  per  cent  of 
Alternaria  rots  in  those  lemons  from  which  the  buttons  had  been 
removed  and  57  per  cent  in  the  checks. 

The  results  recorded  in  the  two  preceding  paragraphs  indicate  that 
it  would  be  impracticable  to  attempt  to  control  Alternaria  rot  in 
lemons  by  sterilization,  even  after  the  buttons  have  been  removed ;  at 
least  this  is  true  for  the  two  sterilizing  solutions  used  in  this  test. 


ALTERNARIA   IN  THE   LEMON   TISSUE    UNDER   THE    BUTTONS 

The  object  of  this  test  was  to  determine  the  percentage  of  fruit 
infection  after  different  periods  of  storage  in  different  packing  houses. 
The  lemons  chosen  were  green  or  silver  when  picked.  Some  of  them 
were  tested  at  once  but  most  of  them  had  been  kept  in  storage  for 
from  one  to  three  months  (see  table  6).  In  order  to  reduce  to  a 
minimum  the  possibility  of  error  through  contamination,  the  different 
lots  of  lemons  were  immersed  for  five  to  ten  minutes  in  mercuric 


Bull.  408] 


ALTERNARIA    ROT    OF    LEMONS 


19 


chloride  solution  at  115°  to  120°  F.  After  drying",  the  stem  end  of 
each  lemon  was  sliced  off  just  below  the  juncture  of  button  and  fruit. 
A  cork  borer  was  then  inserted  into  the  lemon  so  as  to  include  the 
tissues  just  below  the  point  of  attachment  of  the  button.  A  second 
cut  was  now  made,  parallel  to  the  first,  thus  freeing  a  single  disc  of 
tissue  from  each  lemon,  approximately  %6  inch  thick  and  ^  inch  in 
diameter.  All  operations  were  performed  in  an  inoculation  cage  and 
with  sterilized  instruments.  The  discs  of  tissue,  thirty  from  each  lot  of 
lemons,  were  cultured  on  an  artificial  medium  in  Petri  dishes  and 
incubated  at  77°  F.  The  percentages  of  discs  from  each  lot  of  lemons 
which  showed  infection  with  Alternaria  are  shown  in  table  6. 


TABLE  6 

Development  of  Alternaria  from  Discs  of  Tissue  Taken  from  Beneath  the 
Buttons  of  Lemons  that  had  just  been  Picked  or  had  been  Kept  in 
Storage. 


Color  of  lemons  when  picked 

Packing 
house 

Days  lemons  were 

held  in  storage  before 

discs  were  cut 

Percentage  of  discs 

from  which  Alternaria 

developed 

Green 

(          3 

1 

2 

4 
< 

3 

2 

4 

1 

56 
61 
61 
64 
84 
92 
93 
99 

3.3 
6.7 
6.7 
0.0 

10.0 
0.0 
0.0 
3.3 

Silver 

6 

7 

8 

10 

11 

10 

I 

9 
8 
11 
3 
4 
5 

2 

2 

2 

2 

2 
22 
40  ■ 
49 
50 
55 
55 
59 
60 
75 

0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
33.3 

0.0 
3.3 
0.0 

50.0 
0.0 
6.7 

70.0 

The  results  of  this  test  may  be  summarized  as  follows : 
1.  Of  the  240  discs  taken  from  lemons  which  were  green  when 
picked,  an  average  of  only  3.8  per  cent  developed  a  growth  of  Alter- 


20  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

naria.    Some  of  the  lots  that  had  been  stored  for  three  months  showed 
less  decay  than  some  of  those  stored  only  two  months. 

2.  Of  the  270  discs  taken  from  the  nine  lots  of  stored  lemons  which 
were  silver  when  picked,  an  average  of  18.1  per  cent  developed  a 
growth  of  Alternaria. 

3.  Of  the  150  discs  taken  from  the  five  lots  of  silver  lemons  two 
days  after  picking  none  showed  infection. 

These  data  would  indicate  (a)  that  within  certain  limits  (see 
table  2)  the  higher  the  temperature  the  sooner  the  Alternaria  fungus 
will  pass  from  the  buttons  into  the  lemon  tissues  and  cause  them  to 
decay,  (b)  that  the  more  mature  the  fruit  when  picked  the  sooner  it 
will  become  decayed,  (c)  that  the  general  vitality  of  the  fruit  is  a  very 
important  factor  (e.g.,  see  table  6,  packing  house  3,  which  shows  33.3 
per  cent  infection  in  the  silvers  held  40  days  and  no  infection  in  those 
held  59  days),  and  (d)  that  the  Alternaria  fungus  does  not  usually 
pass  from  the  buttons  into  the  silver  and  green  lemons  until  after 
they  have  been  picked  and  have  begun  to  lose  their  vitality. 

The  relations  mentioned  in  (d)  of  the  preceding  paragraph  do  not 
hold  true  for  lemons  that  have  become  tree-ripe  before  picking. 
Several  tests  similar  to  the  preceding  one,  have  shown  that  in  a  com- 
paratively large  number  of  cases  the  fungus  has  passed  from  the 
button  into  the  tissues  beneath  by  the  time  the  tree-ripe  lemons  are 
picked.    This  is  especially  true  during  the  warmer  months. 


STERILIZATION    OF    LEMON    BUTTONS 

Buttons  from  mature  lemons. — The  tests  already  reported  in  this 
paper  have  indicated  that  immersing  the  lemons,  either  with  or  with- 
out the  buttons,  in  mercuric  chloride  or  copper  sulphate  solutions  of 
the  strengths  commonly  used,  does  not  control  Alternaria  rot.  For 
this  reason  an  effort  was  made  to  find  some  method  of  sterilizing 
detached  buttons,  since  in  this  way  much  larger  numbers  could  be 
tested  and  the  results  much  more  quickly  determined.  It  was  hoped 
that  by  this  method  some  medium  might  be  discovered  which  could 
safely  be  used  to  sterilize  the  buttons  while  they  were  still  attached  to 
the  lemons. 

The  methods  employed  in  this  test  are  indicated  in  table  7,  which 
is  a  general  summary  of  the  results  of  many  trials,  with  most  of  the 
details  omitted.  Where  HgCl2  was  used  the  strength  of  the  solution 
was  always  1  to  1000. 


Bull.  408] 


ALTERNARIA   ROT    OF   LEMONS 


21 


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22  UNIVERSITY   OF    CALIFORNIA EXPERIMENT    STATION 

The  conclusions  may  be  summarized  as  follows : 

1.  None  of  the  solutions  used  can  be  considered  to  be  efficient  for 
the  prevention  of  the  development  of  Alternaria  in  detached  lemon 
buttons.  Higher  concentrations,  higher  temperatures  and  longer 
periods  of  exposure  would  doubtless  effect  greater  degrees  of  steriliza- 
tion of  the  detached  buttons,  but  these  would  have  to  be  so  great  that 
the  treatment  would  be  impracticable  under  packing-house  conditions 
and,  furthermore,  would  result  in  injury  to  the  fruit. 

2.  Some  of  the  solutions  appeared  to  have  more  of  a  retarding 
effect  on  Alternaria  than  on  some  of  the  other  organisms  such  as 
Mucor,  Penicillium,  Aspergillus,  Colletotrichum  and  bacteria,  and  as 
a  result  the  largest  percentage  of  infection  is  shown  in  the  column 
headed  "Miscellaneous"  (see  lots  number  12  and  21).  That  this  effect 
was  due  to  retardation  rather  than  killing  of  the  Alternaria  is  at  least 
indicated  by  the  results  from  lots  1  and  2  in  which  there  was  a  marked 
reduction  in  the  percentage  of  Alternaria  but  no  sterilization  in 
lot  1  and  only  7  per  cent  in  lot  2.  Similarly,  in  a  preceding  test  hot 
water  alone  was  used  on  pure  cultures  of  Alternaria  and  the  treat- 
ment failed  to  kill  the  fungus  but  produced  a  very  noticeable  retarda- 
tion in  its  development.  From  these  results  it  appears  that  the 
Alternaria  in  such  cases  as  lots  1,  2,  12  and  21  was  not  actually  killed 
but  that  its  rate  of  growth  was  temporarily  inhibited  or  retarded  to 
such  an  extent  that  it  was  overgrown  and  further  retarded  by  some 
of  the  other  organisms,  and  could  not  be  detected  when  the  cultures 
were  examined.  Repeated  tests  have  shown  that  hot  water  is  not  a 
good  sterilizing  medium  for  the  control  of  Alternaria  rot,  since  its 
retarding  effect  is  apparently  only  temporary. 

3.  An  examination  of  the  data  pertaining  to  the  checks  shows  that 
a  very  large  percentage  of  the  buttons  was  infected  with  Alternaria. 
It  seems  very  probable  that  many  of  those  listed  in  the  miscellaneous 
column  were  infected  with  Alternaria  but  that  it  was  outgrown  by 
other  organisms.  In  lot  5,  for  example,  44  per  cent  of  the  buttons  are 
listed  in  the  miscellaneous  column.  These  buttons  were  taken  from 
lemons  which,  while  practically  mature,  were  on  the  trees  during  the 
freeze  of  1923.  These  particular  lemons,  when  picked  a  few  weeks 
later,  showed  very  slight,  if  any,  visible  injury,  but  it  is  of  interest  to 
note  that  in  the  checks,  44  per  cent  of  the  buttons  from  these  lemons 
showed  a  predominant  growth  of  Colletotrichum.  Apparently  the  low 
temperature  had  produced  changes  in  the  tissues  that  made  them  more 
favorable  for  the  growth  of  Colletotrichum  than  for  that  of  Alter- 
naria. This  possibility  is  also  proved  by  the  fact  that  much  more 
Colletotrichum  rot  is  found  in  lemons  following  a  freeze  than  is  found 


Bull.  408] 


ALTERNARIA    ROT    OP    LEMONS 


23 


under  the  usual  conditions.  With  further  reference  to  lot  5,  it  would 
seem  that  mercuric  chloride,  as  here  used,  is  a  better  sterilizing  agent 
for  Colletotrichum  than  for  Alternaria, 

The  buttons  from  green  and  silver  lemons  are  not  reported 
separately  in  table  7  since  it  appears  that  the  vitality  of  the  tissues 
rather  than  their  age  is  the  principal  factor  determining  the  per- 
centage of  infection.  Apparently  the  vitality  of  the  tissues  governs 
the  depth  of  penetration  of  the  Alternaria  and  in  turn  this  factor  at 
least  partially  determines  the  percentage  of  sterilization. 

Buttons  from  young  lemons. — In  February  buttons  were  taken 
from  immature  lemons,  of  the  average  diameters  shown  in  table  8. 
Before  the  buttons  were  placed  in  the  culture  dishes  they  were  soaked 
in  mercuric  chloride  solution  of  the  usual  strength  for  two  minutes 
at  65°  F. 

TABLE  8 

Percentages  of  Alternaria,  or  Other  Infection,  Found  in  Buttons  Taken 
from  Young  Lemons  of  Different  Sizes. 


Number  of  buttons 

Average  diameter 
of  lemons 

Alternaria 
infection 

Miscellaneous 
infection 

Sterile 

50 
50 
50 

Inches 

l12/l6 

lVl6 

Vl6 

Per  cent 

80 
84 
70 

Per  cent 

8 

6 

24 

Per  cent 

12 

10 

6 

The  data  in  table  8  indicate  that  a  very  high  percentage  of  the 
buttons,  even  from  lemons  that  were  only  %6  of  an  inch  in  diameter, 
were  at  least  potentially  infected  with  Alternaria.  It  should  be  borne 
in  mind,  however,  that  these  buttons  had  been  immersed  in  the 
sterilizing  solution  for  only  two  minutes  and  that  at  least  not  all 
spores  adhering  to  the  surface,  to  say  nothing  of  those  which  may  be 
protected  in  crevices,  are  killed  by  such  an  exposure. 

In  another  test  the  larger  crevices  were  eliminated  by  discarding 
the  enlarged  central  portion  of  the  button,  using  only  the  compara- 
tively thin  outer  layer  of  tissues  which  compose  the  calyx  ring.  The 
sterilizing  conditions  were  also  made  more  rigorous.  The  calyx  rings 
were  first  soaked  for  two  minutes  in  95  per  cent  alcohol,  to  ensure  a 
complete  wetting  of  the  surface,  and  then  for  five  minutes  in  1  part 
mercuric  chloride  to  1000  parts  of  50  per  cent  alcohol.  In  this  test  the 
calyx  rings  from  lemons  of  two  sizes  were  used.  The  larger  lemons 
were  approximately  l1/^  inches  in  diameter  while  the  smaller  ones  were 
picked  just  after  the  petals  had  fallen  and  were  approximately  %  inch 
in  diameter. 


24  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

The  results  of  this  test  showed  90  per  cent  Alternaria  infection  in 
the  calyx  rings  from  the  larger  lemons  and  none  in  those  from  the 
smaller  ones.  In  the  case  of  the  older  calyx  rings  this  seems  to  indi- 
cate either  that  the  sterilizing  agent  did  not  kill  all  surface  spores  or 
that  the  fungus  had  entered  the  tissues  sufficiently  far  to  be  out  of 
reach  of  the  sterilizing  solution  as  applied.  The  results  of  a  micro- 
scopical study  which  will  be  reported  later  in  this  paper,  indicate  that 
the  latter  explanation  is  the  more  probable.  This  conclusion  is  also 
confirmed  by  the  fact  that  the  controls  for  the  smaller  sizes  showed 
60  per  cent  Alternaria  growth  while  those  which  had  been  immersed 
in  the  sterilizing  solution  showed  none.  This  result  indicates  that  the 
treatment  was  sufficient  to  kill  surface-adhering  spores.  Some  por- 
tions of  the  calyx  rings  from  some  of  the  smaller  lemons  had  begun 
to  turn  brown  as  if  infection  had  started,  but  if  this  was  the  case  the 
penetration  of  the  mycelium  was  so  nearly  superficial  that  its  further 
development  was  prevented  by  the  sterilization  process. 

The  high  percentage  of  negative  results  obtained  in  attempting  to 
sterilize  detached  lemon  buttons  seem  to  indicate  that  attempted 
sterilization  of  attached  buttons,  without  injury  to  the  fruit,  is 
impracticable. 


SPRAYING    TO    CONTROL    ALTERNARIA    ROT 

Experiments  were  conducted  with  a  view  to  the  control  of  Alter- 
naria rot  by  means  of  orchard  spraying.  In  one  grove  twelve  rows  of 
twenty  trees  each  were  sprayed  with  Bordeaux  (3-3-50)  in  April 
just  following  the  heaviest  spring  set  of  fruits.  Six  of  these  rows 
were  sprayed  again  in  September  following  the  heaviest  fall  set.  In 
a  second  grove  147  trees  were  sprayed  in  April,  72  of  which  were 
sprayed  again  in  November. 

Cultures  of  buttons  taken  from  the  sprayed  trees  failed  to  reveal 
any  beneficial  effect  from  the  treatment. 

During  the  course  of  the  preceding  spraying  test  a  second  and 
more  carefully  conducted  one  was  made  in  a  different  grove.  Seven 
hundred  twenty  lemon  blossoms  that  had  just  opened  and  500  young 
lemons  about  %  inch  in  diameter,  were  individually  sprayed  with 
Bordeaux  mixture  (4-4-50)  by  a  hand  atomizer.  Care  was  taken  to 
see  that  all  parts  of  the  blossoms,  the  small  fruits,  the  calyx  cups  and 
the  adjacent  p?rts  of  the  stems  were  thoroughly  wet  by  the  spray 
mixture.  Seventy -seven  days  later  the  sprayed  lemons,  and  controls 
of  a  similar  size,  were  picked  and  the  buttons  removed  and  cultured 
to  determine  the  relative  percentages  of  Alternaria  infection  in  the 


BULL.  408]  ALTERNARIA   ROT    OP   LEMONS  25 

three  lots.  When  picked,  the  lemons  from  the  sprayed  blossoms  had 
attained  an  average  diameter  of  2%2  inch  while  the  fruits  which  were 
!/4  inch  in  diameter  when  sprayed  had  become  1%2  inches  in  diameter. 
Buttons  were  taken  from  only  one  lot  of  controls  and  their  average 
diameter  was  approximately  that  of  the  larger  of  the  two  sprayed  lots. 
The  percentages  of  infection  appearing  in  the  three  lots  of  buttons 
were  (a)  in  those  taken  from  the  fruits  from  the  sprayed  blossoms, 
57  per  cent  Alternaria,  43  per  cent  miscellaneous  infection  and  none 
sterile,  ( b )  in  those  from  the  fruits  y±  inch  in  diameter  when  sprayed, 
70  per  cent  Alternaria,  30  per  cent  miscellaneous  and  none  sterile,  and 
(c)  in  the  controls,  68  per  cent  Alternaria,  32  per  cent  miscellaneous 
and  none  sterile. 

The  results  of  the  two  preceding  tests,  coupled  with  the  fact  that 
there  is  an  almost  continuous  setting  of  young  fruits  on  lemon  trees, 
would  indicate  that  there  is  little  hope  of  satisfactorily  controlling  or 
even  materially  reducing  the  percentage  of  Alternaria  rot  in  lemons 
by  means  of  orchard  spraying. 


MICROSCOPICAL     STUDY    OF     THE     LEMON     FRUIT    AND     BUTTON 
TISSUES    INFECTED    WITH    ALTERNARIA 

The  results  of  the  tests  reported  thus  far  in  this  paper  show  the 
comparative  inefficiency  of  the  usual  methods  as  means  of  controlling 
Alternaria  rot  of  lemons.  The  inefficiency  of  these  methods  suggests 
that  spores  or  resistant  mycelium  were  protected  from  the  sterilizing 
solutions  by  being  in  depressions  or  crevices  in  the  button  or  in  the 
fruit  beneath  it,  or  that  the  mycelium  had  already  penetrated  the 
tissues  of  the  button  or  lemon  and  was  out  of  reach  of  the  sterilizing 
solutions.  To  determine  whether  such  could  be  the  case,  microscopical 
studies  were  made  of  the  tissues  in  question.  For  this  purpose  lemon 
buttons  and  portions  of  the  lemon  tissues  just  beneath  the  buttons 
were  collected,  killed,  fixed,  embedded,  sectioned,  stained  with  differ- 
ential stains,  and  the  sections  studied  microscopically  to  detect  the 
presence  or  absence  of  spores  or  mycelium  on  or  in  these  tissues. 

Weak  chrom-acetic  acid  seemed  to  give  better  results  than  any  of 
the  several  other  killing  solutions  tried.  Of  the  several  different  stain 
combinations  commonly  used  for  differentiating  fungus  mycelium  and 
host  tissues,  such  as  Pianeze,  Delaneld's  Haematoxylin  and  Eosin,  and 
Mazdala  Red  and  Light  Green,  the  last  gave  the  best  results. 

The  age  of  the  lemons  from  which  the  buttons  and  other  tissues 
were  taken  is  indicated  by  the  fact  that  all  of  them  had  been  picked 
previously  for  commercial  purposes.    The  color  of  the  lemons  at  time 


26 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


of  picking  and  their  treatment  during  the  interval  between  picking 
and  the  time  at  which  the  buttons  and  lemon  tissues  were  selected  for 
this  study  is  stated  in  the  legends  of  plates  2  and  3.9  Figures  1  to  12, 
plates  2  and  3,  are  photomicrographs  of  portions  of  some  of  the  sec- 
tions of  the  stained  tissues.  The  magnifications  were  the  same  for  all 
of  these  figures. 


Fig.  1. — Outline  of  a  projection  of  a  microscopical  section  which  shows  the 
configuration  of  a  button  and  its  attachment  to  the  fruit.  The  numerals  placed 
on  different  portions  of  the  outline  designate  the  approximate  regions  of  the 
buttons  or  fruits  from  which  the  sections  were  taken  which  are  figured  in 
plates  2  and  3. 


The  microscopical  study  of  the  tissues  in  question  indicates : 

1.  The  ineffectiveness  of  sterilizing  agents  seems  to  be  due  to  the 
presence  of  spores  and  mycelium  in  depressions  or  crevices  where  they 
are  inaccessible  to  the  sterilizing  solution,  and  to  penetration  of 
mycelium  into  the  button  tissues  where  it  is  out  of  reach  of  the 
sterilizing  solution  (pi.  2,  fig.  4,  and  pi.  3,  figs.  5,  6,  7,  and  8). 

2.  In  some  cases  spores  and  resistant  mycelium  were  found  attached 
to  the  surface  of  the  button  but  with  no  evident  attempt  at  penetration 
(pi.  2,  fig.  2).  In  other  cases  apparently  the  spore  had  germinated 
on  the  surface  and  had  made  an  unsuccessful  attempt  at  penetration, 
but  had  produced  certain  changes  in  the  button  tissue  directly  beneath 
(pi.  2,  fig.  1).  In  most  cases  where  the  fungus  mycelium  had  gained 
entrance  the  tissue  of  the  button  had  formed  a,  resistant  or  semi- 
resistant  layer  of  tissue  in  advance  of  the  invasion  of  the  fungus 
(pi.  2,  figs.  1,  3,  and  4,  and  pi.  3,  figs.  6  and  7).  The  cells  composing 
the  resistant  layer  are  or  at  least  very  closely  resemble  young  cork 
cells.  They  are  almost  or  entirely  free  from  protoplasmic  contents 
and  their  exterior  walls  are  thicker  than  their  lateral  or  inner  walls. 


9  To  visualize  the  region  of  the  buttons  or  lemons  from  which  figures  1  to  12, 
plates  2  and  3,  were  taken,  frequent  reference  should  be  made  to  text  figure  1. 


BULL.  408]  ALTERNARIA   ROT    OF   LEMONS  27 

A  cork  cambium  is  very  evident  in  most  cases  and  some  of  the  pre- 
existing cells  which  were  pushed  off  by  the  newly-formed  cork  layers 
are  found  intermingled  with  the  mats  of  spores  and  mycelium  on  the 
surface  (pi.  2,  figs.  3,  and  4,  and  pi.  3,  fig.  7).  In  some  cases  the 
fungus  seems  to  have  been  able  to  pass  this  barrier  imposed  by  the 
host,  and  as  many  as  three  successive  layers  of  cork  tissue  have  been 
formed  at  intervals  in  the  effort  to  check  the  invasion  of  the  fungus. 

3.  In  some  cases  the  invasion  of  the  fungus  was  so  rapid  that 
apparently  the  host  tissues  did  not  have  time  to  form  the  resistant 
layer  of  cork  cells  until  considerable  progress  had  been  made  by  the 
fungus.  Plate  3,  figure  5  shows  such  a  case  which  occurred  under 
field  conditions  (compare  pi.  2,  figs.  1  and  3,  and  pi.  3,  figs.  6  and  7). 
Under  field  conditions  the  vitality  of  the  tissues  of  the  button  and 
lemon  fruit  is  usually  at  its  maximum  while  conditions  for  the 
germination  and  growth  of  the  fungus  are  usually  unfavorable.  But 
when  the  lemons  have  been  picked  and  stored  the  conditions  are 
reversed  and  the  fungus  makes  much  more  rapid  progress.  The  result 
of  an  exaggerated  case  of  this  reversal  of  conditions  is  shown  in  plate  3, 
figures  9  and  12.  In  this  case  buttons  from  green  lemons  were  placed 
in  Petri  dishes  containing  an  artificial  medium  very  favorable  for  the 
growth  of  the  fungus.  Had  these  lemons,  with  their  buttons  attached, 
been  placed  under  usual  storage  conditions  it  probably  would  have  been 
at  least  three  months  before  the  fungus  would  have  progressed  through 
the  buttons  and  into  the  lemons  to  such  an  extent  as  to  cause  visible 
signs  of  decay.  Under  the  favorable  conditions  in  the  Petri  dish  the 
fungus  penetrated  all  portions  of  the  buttons  in  eight  days  and  so 
modified  their  internal  structure  that  the  individual  cells  had  lost 
their  identity,  except  in  the  case  of  the  more  resistant  wood  cells.  In 
the  prepared  sections  the  interior  of  the  buttons  appeared  to  be  almost 
a  homogeneous  mass  with  the  mycelium  penetrating  all  portions  of  it 
(pi.  3,  fig.  12).  The  figure  just  mentioned  should  be  compared  with 
figure  11  in  the  same  plate,  noting  comparative  diameters  of  the 
mycelial  threads  and  the  nature  of  the  host  tissues.  Figure  11  repre- 
sents the  conditions  in  a  button  that  had  been  taken  from  a  lemon 
kept  in  the  sweatroom  for  ten  days  and  then  held  in  storage  for 
another  ten  days. 

4.  As  a  rule  the  fungus  mycelium  has  not  passed  from  the  button 
down  into  the  fruit  tissues  at  the  time  of  picking.  Exceptions  to  this 
rule  may  be  found  in  cases  where,  the  lemons  have  become  over-ripe 
before  picking  or  where  they  have  been  exposed  to  excessively  high  or 
low  temperatures.  Apparently  the  penetration  does  not  take  place 
until  the  lemon  tissues  have  become  "weakened"   (changed  physio- 


28  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

logically)  to  a  certain  extent.  It  seems  also  that  any  environment 
which  produces  a  weakened  condition  in  the  fruit,  such  as  a  com- 
paratively high  storage  temperature,  also  produces  conditions  favor- 
able for  rapid  growth  of  the  fungus.  The  combination  of  these  two 
effects  results  in  a  more  or  less  rapid  decay  of  the  fruit. 

5.  The  microscopical  study  further  showed  why  very  many  of  the 
stored  lemons  may  be  subject  to  the  attack  of  Alternaria  even  though 
the  buttons  have  been  removed  and  the  lemons  soaked  in  a  sterilizing 
solution  before  being  placed  in  storage.  Those  who  are  familiar  with 
the  early  stages  of  development  of  the  lemon  fruit  know  that  after 
the  petals  have  fallen  there  is  left  behind  the  calyx  cup,  in  the  center 
of  which  the  young  fruit  is  attached.  This  calyx  cup  makes  a  suitable 
receptacle  for  catching  Alternaria  spores.  As  the  fruit  enlarges,  the 
edges  of  the  calyx  cup  come  in  contact  with  the  fruit  and  the  Alter- 
naria spores  are  thus  entrapped.  In  this  manner  some  of  the  spores,  or 
bits  of  resistant  mycelium  from  spores  that  germinated  and  grew  for 
only  a  short  time,  may  easily  find  lodgment  in  some  of  the  minute 
depressions  or  crevices  in  the  fruit  under  the  button.  Again,  as  the 
fruit  enlarges  it  no  longer  maintains  an  upright  position  but  hangs 
downward  and  in  many  cases  portions  of  the  edges  of  the  button  lose 
their  close  contact  with  the  fruit.  This  condition  affords  another 
chance  for  the  entrance  of  spores  into  the  space  between  the  button 
and  fruit.  These  spores  or  resistant  bits  of  mycelium  are  not  reached 
by  the  sterilizing  solutions  under  the  usual  methods  of  application. 
Plate  3,  figure  8  shows  two  small  pieces  of  resistant  mycelium  in  a 
microscopical  crevice  in  the  surface  of  a  lemon  just  beneath  the 
button. 

6.  In  describing  the  characteristic  appearance  of  the  lemon  tissues 
beneath  the  button,  in  the  early  stages  of  infection  with  Alternaria,  it 
was  stated  in  a  previous  section  of  this  paper  that  the  individual 
vascular  bundles  appeared  to  be  infected  first,  as  indicated  by  the  fact 
that  they  are  first  to  show  discoloration.  The  microscopical  study  has 
shown  that  initial  infection  probably  takes  place  first  in  the  phloem, 
or  in  the  bundle  sheath,  and  passes  from  there  into  the  surrounding 
parenchyma  or  into  and  among  the  water-conducting  vessels  and  other 
xylem  elements.  Plate  3,  figure  10  shows  mycelium  among  the  wood 
cells  and  medullary  rays  and  in  a  water  conducting  vessel.  No  evi- 
dence was  found  to  indicate  that  the  Alternaria  mycelium  enters  the 
water-conducting  vessels  of  the  button,  while  the  lemon  is  still  attached 
to  the  tree,  to  such  an  extent  as  to  restrict  the  passage  of  water. 


BULL.  408]  ALTERNARIA   ROT    OF   LEMONS  29 


DISCUSSION    OF    CONTROL    MEASURES 

The  commonly  used  methods  of  spraying  and  sterilization  appear 
to  be  in  a  large  measure  ineffective  as  a  means  of  controlling  Alter- 
naria  rot  of  lemons.  The  ineffectiveness  in  the  case  of  spraying  is  very 
largely  due  to  the  fact  that  new  fruits  are  being  formed  almost  con- 
tinuously and  that  Alternaria  spores  are  always  present  in  the  air 
and  on  all  parts  of  the  tree.  These  spores  are  constantly  being  carried 
to  the  newly  formed  tissues  either  by  air  currents  or  rain.  The 
difficulty  in  sterilizing  the  lemon  fruits  after  they  have  been  picked 
lies  in  the  fact  that  the  mycelium  has  already  entered  the  button  tissues 
or  that  the  spores  are  located  in  crevices  in  the  button  or  in  the  lemon 
tissue  beneath  the  button,  so  that  both  mycelium  and  spores  are 
inaccessible  to  the  sterilizing  solutions  commonly  used  for  such  pur- 
poses. Various  new  solutions  are  being  tested  by  different  workers 
and  it  is  hoped  that  one  may  be  found  which-  will  be  able  to  effect 
sterilization  without  injury  to  the  fruit. 

Rogers  and  Earle,10  who  worked  at  San  Pedro,  Isle  of  Pines,  on 
control  measures  for  stem-end  rot11  of  several  types  of  citrus  fruits, 
recommend  that  the  fruits  be  pulled  instead  of  clipped  from  the  tree 
and  that  the  scar  on  the  fruit,  left  by  detaching  it  from  the  stem,  be 
covered  with  shellac.  An  initial  test  of  this  method  proved  it  to  be 
so  inefficient  for  the  control  of  Alternaria  rot  that  it  was  not  given 
further  attention.  Even  were  this  method  more  or  less  effective  it  does 
not  seem  that  it  would  be  practicable  under  California  conditions 
where  such  a  large  crop  of  lemons  is  produced.  Winston,  Fulton  and 
Bowman,12  working  in  Florida,  have  found  that  both  orchard  spraying 
and  the  removal  of  the  buttons  from  the  fruits  very  noticeably  reduce 
the  amount  of  stem-end  rot  in  oranges  and  grapefruit.  They  have 
found  also  that  pruning  the  dead  branches  out  of  the  trees  tends  to 
reduce  stem-end  rot,  especially  where  Diplodia  is  concerned.  The 
almost  universal  presence  of  Alternaria  spores  on  decaying  vegetation, 
coupled  with  the  fact  that  the  new  fruits  are  forming  almost  con- 
tinuously, indicates  that  pruning  would  be  comparatively  ineffective 
in  controlling  Alternaria  rot  of  lemons. 


10  Bogers,  J.  M.,  and  F.  S.  Earle.  A  simple  and  effective  method  of  protect- 
ing citrus  fruits  against  stem-end  rot.     Phytopathology  7:  361-367.     1917. 

11  Stem-end  rot  is  somewhat  similar  to  Alternaria  rot  in  that  the  fungus 
enters  the  fruit  through  the  stem  end.  It  is  principally  caused  by  Phomopsis 
citri  and  Diplodia  natalensis. 

12  Winston,  J.  E.,  H.  E.  Fulton,  and  J.  J.  Bowman.  Commercial  control  of 
stem-end  rot.     The  Florida  Grower  28:  6,  26.     1923. 


30  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

With  reference  to  the  removal  of  the  buttons  as  a  means  of  control, 
it  ordinarily  requires  a  week  or  more  to  sweat  green  lemons  to  give 
them  the  desired  color.  Under  such  conditions  the  buttons  remain 
attached  to  the  lemons.  If  the  conditions  were  intensified  enough 
during  this  process  to  cause  the  buttons  to  drop  there  would  be  the 
added  danger  of  injury  to  the  fruit  and  also  the  probability  of  causing 
physiological  changes  in  it  which  would  favor  the  growth  of  Alternaria. 
In  Florida  it  is  stated  that  an  exposure  to  ethylene  gas,  at  a  tem- 
perature of  80°  to  85°  F  and  a  humidity  of  85  to  90  per  cent  for 
thirty-six  hours  will  cause  orange  and  grapefruit  buttons  to  loosen. 

At  the  present  time  and  under  California  conditions  it  is  not 
possible  to  prevent  Alternaria  rot  of  lemons  satisfactorily,  but  it  has 
been  found  that  the  losses  from  this  disease  may  be  materially  reduced 
by  the  adoption  of  the  following  suggestions : 

1.  The  trees  should  be  kept  as  nearly  as  possible  in  a  healthy  condi- 
tion so  that  the  fruit  will  grow  continuously  from  setting  to  maturity. 
Fruits  that  have  experienced  a  set-back,  due  to  lack  of  water  or  other 
cause,  are  weaker  than  if  they  had  grown  continuously  and  hence  are 
more  susceptible  to  attack  by  Alternaria, 

2.  The  fruit  should  be  picked  while  it  has  a  high  vitality,  i.e., 
while  it  is  yet  silver  or  green  in  color.  The  Alternaria  rot  fungus 
cannot  attack  it  as  long  as  its  vitality  is  high. 

3.  Washing  the  fruit  in  hot  water  materially  raises  its  temperature. 
If  this  temperature  could  be  reduced  before  the  fruit  is  placed  in 
storage  the  chances  for  the  development  of  Alternaria  rot  would 
probably  be  reduced.  It  is  a  well-known  fact  that  a  sudden  reduction 
of  temperature,  following  a  comparatively  high  one,  markedly  retards 
the  development  of  various  diseases, 

4.  If  the  fruit  can  be  dried  sufficiently  to  remove  all  free  water 
from  beneath  the  buttons  before  the  fruit  is  placed  in  the  storeroom, 
conditions  are  made  less  favorable  for  the  development  of  Alternaria. 

5.  Minimum  temperature,  humidity  and  time  of  exposure  should 
be  used  when  sweating  green  fruit. 

6.  So  far  as  possible,  all  fruits  affected  with  endoxerosis  (internal 
decline,  blossom-end  decay,  etc.)  should  be  excluded. 

7.  The  fruit  should  be  stored  at  a  low  temperature.  The  develop- 
ment of  Alternaria  rot  in  lemons  is  extremely  slow  at  a  temperature  of 
55°  F,  or  lower,  unless  the  fruit  has  a  low  vitality. 

8.  During  the  process  of  grading  and  packing,  an  unnecessarily 
long  exposure  of  the  fruit  to  the  warmer  temperature  of  the  grading 
room  should  be  avoided. 


BULL.  408]  ALTERNARIA   ROT    OP    LEMONS  31 

9.  Before  packing  for  shipment  each  lot  of  fruit  should  be  care- 
fully inspected  for  the  presence  of  Alternaria  rot  in  its  early  stages, 
i.e.,  before  it  is  visible  on  the  surface.  This  inspection  should  be  made 
by  slicing  off  the  stem  end  of  the  lemon,  just  under  the  button.  If 
the  lemon  has  become  infected  the  small  bundles  of  tissues  in  the 
center  of  the  cut  surface  will  have  a  pinkish  to  light  brown  color.  If 
a  given  lot  of  fruit  examined  in  this  manner  shows  more  than  an 
extremely  low  percentage  of  infection  it  should  not  be  shipped,  or  if 
shipped  it  should  be  placed  on  a  market  where  the  time  consumed  in 
transit  will  be  short  and  where  the  chances  for  its  early  consumption 
are  good. 

SUMMARY 

1.  Alternaria  probably  causes  more  California  lemon  fruits  to 
decay  than  any  other  one  fungus,  with  the  possible  exception  of 
Penicillium. 

2.  Alternaria  rot  may  be  found  in  all  lemon-growing  districts  of 
California. 

3.  The  lemon  fruits  become  potentially  infected  with  Alternaria 
before  picking,  but  they  do  not  usually  begin  to  decay  until  after 
being  placed  in  storage. 

4.  Alternaria  rot  of  lemons  is  caused  by  the  fungus  Alternaria; 
endoxerosis  of  lemons  is  not  caused  by  a  fungus  but  by  abnormal 
physiological  conditions.  Care  should  be  exercised  not  to  confuse 
the  two. 

5.  The  temperature  range  most  suitable  for  the  maximum  growth 
rate  of  Alternaria  either  in  the  lemon  tissues  or  in  the  culture,  is 
approximately  78°  to  83°  F. 

6.  Isolated  Alternaria  spores  or  resistant  mycelium  may  be  killed 
by  a  two-minute  exposure  to  1 :1000  mercuric  chloride,  but  if  they 
are  in  contact  with  the  surface  of  the  lemons,  buttons,  or  twigs,  it 
appears  that  an  exposure  of  at  least  six  to  eight  minutes  is  required 
in  order  to  kill  them. 

7.  To  attempt  to  prevent  Alternaria  rot  of  lemons  by  the  methods 
commonly  used  for  such  purposes  seems  impracticable. 

8.  The  removal  of  the  buttons  before  placing  the  lemons  in  storage 
is  not  effective  in  controlling  Alternaria  rot,  and,  furthermore,  such 
a  procedure  would  be  impracticable. 

9.  Although  the  button  becomes  infected  while  the  fruit  is  very 
young  and  spores  are  also  entrapped  under  the  button,  it  is  only  in 
exceptional  cases  that  the  Alternaria  fungus  has  entered  the  fruit 
tissues  by  the  time  the  lemons  are  picked. 


32  UNIVERSITY    OP    CALIFORNIA EXPERIMENT    STATION 

10.  Alternaria  may  enter  the  lemon  directly  from  the  button  or 
from  spores  or  mycelium  harbored  between  the  button  and  the  lemon. 

11.  If  lemons  of  average  vitality  are  stored  at  a  temperature  of 
55°  to  60°  F,  Alternaria  rot  will  usually  begin  to  appear  in  tree-ripes 
in  from  one  month  to  six  weeks,  in  silvers  in  from  two  to  two  and  a 
half  months,  and  in  greens  from  three  to  four  months. 

12.  Present  methods  of  orchard  spraying  appear  to  be  both  unsuc- 
cessful and  impracticable  for  the  prevention  of  Alternaria  rot  of 
lemons. 

13.  A  microscopical  study  of  the  button  and  lemon  tissues  con- 
cerned has  shown  (a)  attempts  at  sterilization  were  unsuccessful 
because  spores  and  resistant  mycelium  were  located  in  depressions  or 
crevices  where  they  were  inaccessible  to  the  sterilizing  solution  and 
because  in  most  cases  some  of  the  mycelium  had  already  penetrated 
the  button  tissue  where  it  too  was  out  of  reach  of  the  sterilizing  solu- 
tion, (&)  the  button  tissues  retard  the  advance  of  the  fungus,  after 
it  has  once  entered,  by  the  formation  of  a  corky  layer,  (c)  as  the 
Alternaria  enters  the  fruit  it  first  follows  the  bundle  sheath  and  then 
it  passes  into  the  parenchyma  and  into  the  water-conducting  vessels 
and  other  wood  elements,  and  (d)  crevices  and  depressions  in  the 
lemon  tissues  under  the  button  harbor  spores  and  resistant  bits  of 
mycelium  which,  though  the  button  be  removed,  may  produce  infection 
as  soon  as  conditions  become  favorable. 

14.  Certain  methods  of  picking,  storing,  etc.,  will  materially  reduce 
the  amount  of  loss  due  to  Alternaria  rot. 

It  may  be  said  in  conclusion  that  while  Alternaria  is  the  organism 
which  directly  produces  the  decay  under  discussion  in  this  paper,  it 
is  realized  that  some  of  the  other  organisms,  often  found  associated 
with  the  Alternaria,  may  contribute  toward  making  conditions  more 
favorable  for  its  development. 


ACKNOWLEDGMENTS 

The  author  wishes  to  acknowledge  the  helpful  suggestions  given  by 
Dr.  H.  S.  Fawcett,  the  assistance  given  by  Mr.  C.  0.  Smith  in  making 
and  examining  many  of  the  cultures,  and  the  hearty  cooperation  of 
many  of  the  Lemon  Associations  during  the  course  of  this  investigation. 


PLATE  1 

In  comparing  these  figures  illustrating  the  characteristics  of  endoxerosis 
and  Alternaria  rot  of  lemons,  note  especially  the  initial  points  of  attack,  the 
color  differences,  and  the  conditions  of  the  affected  tissues. 

Fig.  1.  A  green,  but  practically  mature,  lemon  showing  an  early  stage  of 
endoxerosis.  Note  deposits  of  gum  near  stylar  end  of  lemon  and  in  the  peel 
at  one  side,  and  the  more  or  less  typical  precoloring  of  the  surface  of  the  peel 
at  the  stylar  end. 

Figs.  2  and  3.  Two  different  types  of  development  in  the  later  stages  of 
endoxerosis. 

Fig.  4.  A  comparatively  early  stage  in  the  development  of  Alternaria  rot. 
Note  area  of  initial  infection  under  the  button,  also  the  advancing  area  of 
infection  down  the  center  of  the  lemon. 

Fig.  5.  Intermediate  stage  in  the  development  of  Alternaria  rot.  Even  at 
this  stage  it  is  not  possible  to  detect  the  infection  without  cutting  the  lemon. 

Fig.  6.  A  later  stage  of  Alternaria  rot.  Following  this  stage  the  entire 
fruit  becomes  infected  and  becomes  a  dark-brown  to  almost  black,  soft  mass 
of  decaying  tissues. 


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PLATE  2 

To  visualize  the  region  of  the  button  or  fruit  from  which  figures  1  to  12, 
plates  2  and  3,  were  taken,  reference  should  be  made  to  text  figure  1. 

Figs.  A  and  B.  Mats  of  Alternaria  mycelium  growing  from  the  buttons  of 
lemons  that  had  been  stored  where  the  temperature  and  humidity  were  too  high. 

Fig.  1.  From  the  button  of  a  silver  lemon  just  after  picking.  Resistant 
spores  and  hyphae,  below  which  cork  cells  have  formed. 

Fig.  2.  From  the  button  of  a  green  lemon  just  after  picking.  Masses  of 
resistant  spores  adhering  to  the  surface  of  the  button.  The  tissues  below 
apparently  unaffected. 

Fig.  3.  From  the  button  of  a  silver  lemon  just  after  picking.  Masses  of 
resistant  spores  above  the  infected  tissue  which  is  penetrated  by  hyphae.  Note 
resistant  layer  of  cork  cells  below  infected  tissues. 

Fig.  4.  Portion  of  a  crevice  in  a  button  from  a  green  lemon  that  had  been 
in  storage  ten  days.  During  this  period  the  temperature  ranged  from  75°  to 
85°  F  and  the  air  was  almost  saturated  with  moisture.  Note  that  there  are 
hyphae  in  upper  half  of  figure  but  none  below  the  resistant  corky  layer  in  the 
lower  half  of  figure. 


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PLATE  3 

Fig.  5.  From  the  button  of  a  yellow  lemon  just  after  picking.  Clumps  of 
resistant  hyphae  formed  on  the  surface  of  the  infected  tissues  below. 

Fig.  6.  Source,  same  as  for  figure  5.  Eesistant  hyphae  in  the  infected 
tissues.     Note  again  corky  resistant  layer  below. 

Fig.  7.  Source,  same  as  for  figure  5.  Portion  of  a  crevice  containing  spores 
and  hyphae;  their  advance  at  least  retarded  by  the  corky  resistant  layer. 

Fig.  8.  From  a  green  lemon  sweated  ten  days  and  then  held  in  storage 
ten  days  longer.  Enlarged  ends  of  hyphae  in  one  of  the  microscopical  depres- 
sions or  crevices  which  appear  in  the  surface  of  the  lemon  under  the  button. 

Fig.  9.  From  button  of  a  green  lemon  just  picked;  button  removed,  placed 
in  semesan  sterilizing  solution  in  90  per  cent  vacuum  for  one  hour,  then  plated 
on  glucose-potato  agar  for  eight  days.  Note  vigorous  growth  of  fungus  in  the 
tissues  under  these  conditions. 

Fig.  10.  Source,  same  as  for  figure  8.  Hyphae  showing  in  a  tangential 
section  of  the  wood  tissue  of  a  button  from  a  green  lemon. 

Fig.  11.  Source,  same  as  for  figure  8.  Hyphae  in  parenchyma  tissue  of 
lemon;  about  one-quarter  inch  below  button. 

Fig.  12.  Source,  same  as  for  figure  9.  Hyphae  in  cortex  parenchyma  of  the 
lemon  button. 


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